Kentaro Shimokado

Role of Transglutaminase 2 in Liver Injury via Cross-linking and Silencing of Transcription Factor Sp1

BACKGROUND & AIMS Despite high morbidity and mortality of alcoholic liver disease worldwide, the molecular mechanisms underlying alcohol-induced liver cell death are not fully understood. Transglutaminase 2 (TG2) is a cross-linking enzyme implicated in apoptosis. TG2 levels and activity are increased in association with various types of liver injury. However, how TG2 induces hepatic apoptosis is not known. METHODS Human hepatic cells or primary hepatocytes from rats or TG2+/+ and TG2-/- mice were treated with ethanol. Mice were administered anti-Fas antibody or alcohol. Liver sections were prepared from patients with alcoholic steatohepatitis. Changes in TG2 levels, Sp1 cross-linking and its activities, expression of hepatocyte growth factor receptor, c-Met, and hepatic apoptosis were measured. RESULTS Ethanol induced apoptosis in hepatic cells, enhanced activity and nuclear accumulation of TG2 as well as accumulation of cross-linked and inactivated Sp1, and reduced expression of the Sp1-responsive gene, c-Met. These effects were rescued by TG2 knockdown, restoration of functional Sp1, or addition of hepatocyte growth factor, whereas apoptosis was reproduced by Sp1 knockdown or TG2 overexpression. Compared with TG2+/+ mice, TG2-/- mice showed markedly reduced hepatocyte apoptosis and Sp1 cross-linking following ethanol or anti-Fas treatment. Treatment of TG2+/+ mice with the TG2 inhibitors putrescine or cystamine blocked anti-Fas-induced hepatic apoptosis and Sp1 silencing. Moreover, enhanced expression of cross-linked Sp1 and TG2 was evident in hepatocyte nuclei of patients with alcoholic steatohepatitis. CONCLUSIONS TG2 induces hepatocyte apoptosis via Sp1 cross-linking and inactivation, with resultant inhibition of the expression of c-Met required for hepatic cell viability.

Apolipoprotein CIII Links Hyperlipidemia With Vascular Endothelial Cell Dysfunction

Background— Apolipoprotein CIII (apoCIII) is a component of some triglyceride-rich very-low-density and low-density lipoprotein and is elevated in dyslipidemia with insulin resistance and the metabolic syndrome. We previously reported that apoCIII directly activates proinflammatory and atherogenic signaling in vascular endothelial cells through protein kinase C-&bgr; (PKC&bgr;). Because PKC&bgr; impairs the response of vascular endothelial cells to insulin, we tested the hypothesis that apoCIII affects insulin signaling in vascular endothelial cells and its function in vitro and in vivo. Methods and Results— ApoCIII inhibited insulin-induced tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), decreasing phosphatidylinositol 3-kinase (PI3K)/Akt activation in human umbilical vein endothelial cells. These effects of apoCIII led to reduced endothelial nitric oxide synthase (eNOS) activation and NO release into the media. ApoCIII activated PKC&bgr; in human umbilical vein endothelial cells, resulting in IRS-1 dysfunction via serine phosphorylation. ApoCIII also activated mitogen-activated protein kinase through PKC&bgr;. The impaired insulin signaling was restored by PKC&bgr; inhibitor or MEK1 inhibitor. ApoCIII-rich very-low-density lipoprotein and apoCIII impaired insulin signaling in the aorta of C57BL/6J mice and in human umbilical vein endothelial cells, which was recovered by PKC&bgr; inhibitor. They also inhibited endothelium-dependent relaxation of the aortas of C57BL/6J mice. In summary, apoCIII in very-low-density lipoprotein impaired insulin stimulation of NO production by vascular endothelium and induced endothelial dysfunction in vivo. This adverse effect of apoCIII was mediated by its activation of PKC&bgr;, which inhibits the IRS-1/PI3K/Akt/eNOS pathway. Conclusion— Our results suggest that apoCIII is a crucial link between dyslipidemia and insulin resistance in vascular endothelial cells with consequential deleterious effects on their atheroprotective functions.

Atorvastatin Attenuates Remnant Lipoprotein-Induced Monocyte Adhesion to Vascular Endothelium Under Flow Conditions

Abstract— Remnant lipoproteins have been reported to play a causative role in atherogenesis. We investigated the effect of remnant-like lipoprotein particles (RLPs) on monocyte-endothelial interaction and their potential regulation by atorvastatin. Monocytic U937 cells were incubated with RLPs isolated from hypertriglyceridemia subjects and their adhesion to human umbilical vein endothelial cells (HUVECs) was examined under flow conditions. Incubation of U937 cells with 15 &mgr;g protein/mL RLPs increased their adhesion to HUVECs activated with IL-1&bgr; (untreated: 6.8±1.6 cells/HPF versus RLPs: 16.2±3.3 cells/HPF, P <0.05). Flow cytometric analysis revealed that incubation with RLPs increased expression levels of CD11a, CD18, and CD49d in U937 cells. Moreover, RLP-induced RhoA activation as well as FAK activation was seen in U937 cells, and RLP-induced RhoA activation seemed to be involved with PKC-dependent signaling. To explore the effect of atorvastatin on RLP-induced U937 cell adhesion to HUVECs, U937 cells were incubated with RLPs in the presence of atorvastatin. Pretreatment of U937 cells with 10 &mgr;mol/L atorvastatin significantly decreased RLP-induced U937 cell adhesion to activated HUVECs (RLP 15.2±1.5 cells/HPF versus atorvastatin+RLP 10.2±1.0 cells/HPF;P <0.05) and decreased the enhanced integrin expression in RLP-treated U937 cells. Atorvastatin also inhibited RLP-induced RhoA activation and FAK activation in U937 cells. In summary, RLPs induced monocyte adhesion to vascular endothelium by sequential activation of PKC, RhoA, FAK, and integrins, indicating a role of remnant lipoproteins in vascular inflammation during atherogenesis. Atorvastatin attenuated this enhanced monocyte adhesion to HUVECs, suggesting an antiinflammatory role for this compound.

Toll-Like Receptor 2 Mediates Apolipoprotein CIII–Induced Monocyte Activation: Retracted

Apolipoprotein (apo)CIII predicts risk for coronary heart disease. We recently reported that apoCIII directly activates human monocytes. Recent evidence indicates that toll-like receptor (TLR)2 can contribute to atherogenesis through transduction of inflammatory signals. Here, we tested the hypothesis that apoCIII activates human monocytoid THP-1 cells through TLR2. ApoCIII induced the association of TLR2 with myeloid differentiation factor 88, activated nuclear factor (NF)-&kgr;B in THP-1 cells, and increased their adhesion to human umbilical vein endothelial cells (HUVECs). Anti-TLR2 blocking antibody, but not anti-TLR4 blocking antibody or isotype-matched IgG, inhibited these processes (P<0.05). ApoCIII bound with high affinity to human recombinant TLR2 protein and showed a significantly higher (P<0.05) and saturable binding to 293 cells overexpressing human TLR2 than to parental 293 cells with no endogenous TLR2. Overexpression of TLR2 in 293 cells augmented apoCIII-induced NF-&kgr;B activation and &bgr;1 integrin expression, processes inhibited by anti-apoCIII antibody as well as anti-TLR2 antibody. Exposure of peripheral blood monocytes isolated from C57BL/6 (wild-type) mice to apoCIII activated their NF-&kgr;B and increased their adhesiveness to HUVECs. In contrast, apoCIII did not activate monocytes from TLR2-deficient mice. Finally, intravenous administration to C57BL/6 mice of apoCIII-rich very-low-density lipoprotein (VLDL), but not of apoCIII-deficient VLDL, activated monocytes and increased their adhesiveness to HUVECs, processes attenuated by anti-TLR2 or anti-apoCIII antibody. ApoCIII-rich VLDL did not activate monocytes from TLR2-deficient mice. In conclusion, apoCIII activated monocytes at least partly through a TLR2-dependent pathway. The present study identifies a novel mechanism for proinflammatory and proatherogenic effects of apoCIII and a role for TLR2 in atherosclerosis induced by atherogenic lipoproteins.

Japan as the front‐runner of super‐aged societies: Perspectives from medicine and medical care in Japan

The demographic structure of a country changes dramatically with increasing trends toward general population aging and declining birth rates. In Japan, the percentage of the elderly population (aged ≥65 years) reached 25% in 2013; it is expected to exceed 30% in 2025 and reach 39.9% in 2060. The national total population has been decreasing steadily since its peak reached in 2008, and it is expected to fall to the order of 80 million in 2060. Of the total population, those aged ≥75 years accounted for 12.3% as of 2013, and this is expected to reach 26.9% in 2060. As the demographic structure changes, the disease structure changes, and therefore the medical care demand changes. To accommodate the medical care demand changes, it is necessary to secure a system for providing medical care. Japan has thus far attained remarkable achievements in medical care, seeking a better prognosis for survival; however, its medical care demand is anticipated to change both qualitatively and quantitatively. As diseases in the elderly, particularly in the old‐old population, are often intractable, conventional medical care must be upgraded to one suitable for an aged society. What is required to this end is a shift from “cure‐seeking medical care” focusing on disease treatment on an organ‐specific basis to “cure and support‐seeking medical care” with treatments reprioritized to maximize the quality of life (QOL) for the patient, or a change from “hospital‐centered medical care” to “community‐oriented medical care” in correlation with nursing care and welfare.

Phosphatidylinositol 3-Kinase Is Required for Growth Factor–Induced Amino Acid Uptake by Vascular Smooth Muscle Cells

Although accumulating evidence suggests that phosphatidylinositol 3-kinase (PI3K) is a common signaling molecule for growth factor-induced amino acid uptake by the cell, the role of PI3K in the uptake of different amino acids was not tested under the same conditions. In this study, we asked whether PI3K mediates platelet-derived growth factor (PDGF) -stimulated uptake of different amino acids that are taken up through 3 major amino acid transporters expressed in rat vascular smooth muscle cells and other cell types and whether PI3K mediates amino acid uptake stimulated with different growth factors and vasoactive substances. PDGF increased the uptake of [(3)H]leucine, [(3)H]proline, and [(3)H]arginine in a dose- and time-dependent fashion. Two different PI3K inhibitors, wortmannin (100 nmol/L) and LY294002 (10 micromol/L), completely inhibited the amino acid uptake stimulated by PDGF. Chinese hamster ovary cells expressing both PDGF receptor-beta and a dominant-negative PI3K did not increase their leucine uptake when stimulated with PDGF, whereas the same cells expressing only PDGF receptor-beta did. Transforming growth factor-beta, as well as insulin-like growth factor-I and angiotensin II, increased leucine uptake by vascular smooth muscle cells. Wortmannin and LY294002 inhibited this increase. We also found that transforming growth factor-beta stimulated PI3K activity and the phosphorylation of Akt, a downstream signaling molecule of PI3K. A similar effect of PI3K inhibitors on amino acid uptake was observed in Swiss 3T3 cells. We conclude that PI3K mediates the uptake of different amino acids by vascular smooth muscle cells and other cell types stimulated with a variety of growth factors, including transforming growth factor-beta. Our findings suggest that PI3K may play an important role in vascular pathophysiology by regulating amino acid uptake.

Concomitant secretion of big endothelin and its C-terminal fragment from human and bovine endothelial cells

A specific radioimmunoassay (RIA) for the carboxyl-terminal fragment (CTF) of big porcine endothelin (pET), an intermediate form of pET, was established to characterize big ET-like and its CTF-like immunoreactivity (LI) secreted from cultured bovine and human endothelial cells (EC). The antibody used crossreacted equally with big pET(1-39) and its CTF(22-39), but not with pET(1-21). Serial dilution curves of the culture media from bovine and human EC were parallel to that of standard CTF. Reverse-phase HPLC coupled with RIAs for big ET and ET of the culture media from bovine and human EC revealed essentially the same elution profiles: two major CTF-LI components, one corresponding to big pET(1-39) and the other to its CTF(22-39), in addition to one major ET-LI component corresponding to pET(1-21). The amounts of CTF-LI were almost equal to that of ET-LI on a molar basis. These data suggest that big ET is processed by a putative ET converting enzyme to yield its CTF and the mature ET(1-21) in EC.

Protein tyrosine kinase inhibitors inhibit chemotaxis of vascular smooth muscle cells.

The effects of protein tyrosine kinase inhibitors on platelet-derived growth factor (PDGF)-induced chemotaxis in cultured rat aortic smooth muscle cells (SMCs) were investigated to elucidate the role of tyrosine phosphorylation in the chemotaxis of vascular SMCs. Two tyrosine kinase inhibitors, methyl 2,5-dihydroxycinnamate and genistein, inhibited PDGF-induced chemotaxis, the IC50 being 5 and 150 mumol/L, respectively. Methyl cinnamate and genistein partly inhibited the adhesion of SMC to collagen-coated dishes. A chemotaxis assay using double-well culture dishes revealed that both agents also inhibited cell migration after adhesion. H-7, a C kinase inhibitor, did not inhibit either chemotaxis or SMC adhesion at 100 mumol/L. Western blot analysis using anti-phosphotyrosine revealed that the tyrosine kinase inhibitors inhibited the tyrosine phosphorylation of at least two proteins of molecular weight 85 and 95 kD under our experimental conditions. An immunocytochemical study revealed that these inhibitors eliminated tyrosine phosphorylation along the cell margins; these agents also inhibited the reorganization of microtubules and stress fibers, both of which are involved in directional cell locomotion. These findings suggest that tyrosine kinases may play an important role in SMC chemotaxis.

VLDL Induces Adipocyte Differentiation in ApoE-Dependent Manner

Objective—To clarify the role of very low density lipoprotein (VLDL) and apolipoprotein E (apoE) in adipogenesis, we studied newly developed hyperlipidemic obese (ob/ob;apoE−/−) mice. Because hydrolysis of VLDL is believed to be the major source of adipogenic free fatty acids, a higher plasma level of VLDL in these mice should exaggerate obesity. Methods and Results—When fed a high-fat, high-cholesterol diet, ob/ob;apoE−/− mice did not show increased body weight or an increased amount of adipose tissue in spite of increased plasma VLDL levels, whereas ob/ob mice showed an increased body weight and amount of adipose tissue, suggesting that there is a novel apoE-dependent pathway for adipogenesis. In vitro experiments using bone marrow stromal cells and 3T3-L1 cells confirmed this notion. ApoE-deficient VLDL did not induce adipogenesis, whereas normal VLDL induced adipogenesis in these cells. The incubation of apoE-deficient VLDL with recombinant human apoE restored its adipogenic activity. Tetrahydrolipstatin, a lipoprotein lipase inhibitor, did not affect the adipogenic activity of VLDL, suggesting that hydrolysis of VLDL did not play a major role in its effects. In fact, lipid components of VLDL or free fatty acids induced only partial adipogenesis. Conclusions—Our findings indicate that VLDL induces adipogenesis in an apoE-dependent manner both in vitro and in vivo.

Pitavastatin Inhibits Remnant Lipoprotein-Induced Macrophage Foam Cell Formation Through ApoB48 Receptor–Dependent Mechanism

Objective— Atherogenic remnant lipoproteins (RLPs) are known to induce foam cell formation in macrophages in vitro and in vivo. We examined the involvement of apoB48 receptor (apoB48R), a novel receptor for RLPs, in that process in vitro and its potential regulation by pitavastatin. Methods and Results— THP-1 macrophages were incubated in the presence of RLPs (20 mg cholesterol/dL, 24 hours) isolated from hypertriglyceridemic subjects. RLPs significantly increased intracellular cholesterol ester (CE) and triglyceride (TG) contents (4.8-fold and 5.8-fold, respectively) in the macrophages. Transfection of THP-1 macrophages with short interfering RNA (siRNA) against apoB48R significantly inhibited RLP-induced TG accumulation by 44%. When THP-1 macrophages were pretreated with pitavastatin (5 &mgr;mol/L, 24 hours), the expression of apoB48R was significantly decreased and RLP-induced TG accumulation was reduced by 56%. ApoB48R siRNA also inhibited TG accumulation in THP-1 macrophage induced by &bgr;–very-low-density lipoprotein derived from apoE−/− mice by 58%, supporting the notion that apoB48R recognizes and takes-up RLPs in an apoE-independent manner. Conclusions— RLPs induce macrophage foam cell formation via apoB48R. Pitavastatin inhibits RLP-induced macrophage foam cell formation. The underlying mechanism involves, at least in part, inhibition of apoB48R-dependent mechanism. Our findings indicate a potential role of apoB48R in atherosclerosis.